Antibodies possess a variety of properties which make them useful as therapeutic molecules. In addition to their ability to bind with high affinity to a molecular target inside or outside of cells with high specificity and selectivity, antibodies render their targeted binding partners susceptible to Fc-receptor cell-mediated phagocytosis and killing through effector functions, such as complement induced pathways and ADCC (antibody-dependent cell-mediated cytotoxicity) related activities.
Further, antibodies may be engineered in a variety of ways to further increase their therapeutic utility. Antibodies having extended in vivo half-lives, for example, may be produced by engineering Fc fusion molecules, by treatment with biocompatible polymers such as polyethylene glycol (PEG), or “pegylation” and by other engineering methods well known in the art. Antibodies have binding specificities for at least two different antigens, called bispecific antibodies (BsAbs), have also been engineered. See Nolan, O. and R. O'Kennedy (1990) Biochim Biophys Acta 1040(1): 1-11.; de Leij, L. et al., Adv Drug Deliv Rev 31(1-2): 105-129 (1998); and Carter, P. J Immunol Methods 248(1-2): 7-15 (2001)). While classical antibodies have identical sequences in each of the two arms (containing the antigen binding sites of Fab region) of the Y-shaped molecule, bispecific antibodies have different sequences in each of the two Fab regions so that each arm of the Y-shaped molecule binds to a different antigen or epitope.
By being able to bind two different antigenic molecules or different epitopes, BsAbs offer a wide variety of clinical applications as targeting agents for in vitro and in vivo diagnostics and immunotherapies. In diagnostic areas, BsAbs have been used, e.g., to study functional properties of cell surface molecules, different Fc receptors and their ability to mediate cytotoxicity (Fanger et al., Crit. Rev. Immunol. 12:101-124 (1992); Nolan et al., Biochem. Biophys. Acta. 1040:1-11 (1990); and to immobilize enzymes and other agents to produce immunodiagnostic and immunoassay reagents and methods.
Bispecific antibodies can also be used for in vitro or in vivo diagnoses of various disease states, including cancer (Songsivilai et al., Clin. Exp. Immunol. 79:315 (1990)). For example, one arm of the BsAb can be engineered to bind a tumor-associated antigen and the other arm to bind a detectable marker. (See, e.g., Le Doussal et al., J. Nucl. Med. 34:1662-1671 (1993), in which a BsAb having one arm which bound a carcinoembryonic antigen (CEA) and another arm which bound DPTA was used for radioimmunodetection of colorectal and thyroid carcinomas. See also Stickney et al., Cancer Res. 51:6650-6655 (1991), describing a strategy for detecting colorectal cancers expressing CEA by radioimmunodetection.
The use of bispecific antibodies for immunotherapy of cancer has been reviewed (see e.g., Nolan and O'Kennedy 1990, supra; de Leij et al. (1998) supra; and Carter, P. (2001) supra.) BsAbs can be used to direct a patient's cellular immune defense mechanisms specifically to a tumor cell or an infectious agent (e.g., virally infected cells such as HIV or influenza virus; protozoa such as Toxoplasma gondii). In particular, one can redirect immune modulated cytotoxicity by engineering one arm of the BsAb to bind to a desired target (e.g. tumor cell or pathogen) and the other arm of the BsAb to bind to a cytotoxic trigger molecule, such as the T-cell receptor or a Fc gamma receptor (thereby activating downstream immune effector pathways). Using this strategy, BsAbs which bind to the Fc gamma RIII have been shown to mediate tumor cell killing by natural killer (NK) cell/large granular lymphocyte (LGL) cells in vitro and to prevent tumor growth in vivo. (See, e.g., Segal et al., Chem. Immunol. 47:179 (1989); Biologic Therapy of Cancer 2(4) DeVita et al. eds. J. B. Lippincott, Philadelphia (1992) p. 1.) In another example, a bispecific antibody having one arm that binds Fc gamma RIII and another that binds the HER2 receptor was developed for treatment of tumors that overexpress HER2 antigen (Hseih-Ma et al. Cancer Research 52:6832-6839 (1992); and Weiner et al. Cancer Research 53:94-100 (1993)). See also Shalaby et al., J. Exp. Med. 175(1):217 (1992) in which a fully humanized F(ab′)2 BsAb comprising anti-CD3 linked to anti-p185(HER2) was used to target T cells to kill tumor cells that overexpress HER2 receptor.
Use of bispecific antibodies has been hindered by difficulties in obtaining BsAbs in sufficient quantity and purity. Traditionally, BsAbs were made using hybrid-hybridoma technology (Millstein and Cuello, Nature 305:537-539 (1983)). Methods for making BsAbs by chemical coupling have since been described (see, e.g., Shalaby et al., J. Exp. Med. 175:217-225 (1992); Rodrigues et al., Int. J. Cancers (Suppl.) 7:45-50 (1992); Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Diabody technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided alternative procedures for making BsAb fragments; as has the use of single chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol. 152: 5368 (1994).
To produce multispecific (e.g., bispecific) antibody heteromultimers (e.g., heterodimers), it is desirable to use methods that favor formation of the desired heteromultimer over homomultimer(s). One method for obtaining Fc-containing BsAbs remains the hybrid hybridoma technique, in which two antibodies are co-expressed (Milstein and Cuello, Nature 305:537-540 (1983); see Suresh, M. R., et al. Methods Enzymol 121:210-228 (1986)). However, it is often inefficient with respect to yield and purity, the desired heteromultimer often being difficult to further purify. Other techniques to favor heteromultimer formation have been described and involve engineering sterically complementary mutations in multimerization domains at the CH3 domain interface, referred to as a “knobs-into-holes” strategy (see e.g., Ridgway et al., Protein Eng. 9:617-621 (1996); Merchant et al., Nat. Biotechnol. 16(7): 677-81 (1998); see also U.S. Pat. Nos. 5,731,168 and 7,183,076). Techniques involving replacing one or more residues that make up the CH3-CH3 interface in both CH3 domains with a charged amino acid for promoting the heterodimer formation have also been described. WO2009/089004.
It would be desirable to find new methods for engineering bispecific antibody fragments and/or full length BsAbs, such as those which enable the BsAbs to be expressed and recovered directly or efficiently from recombinant cell culture and/or which may be produced with efficient yields and purities, or having increased stability compared to bispecific antibodies in the art.